Project Summary Fragile X syndrome (FXS), the most common inherited form of mental retardation, result from mutations that disrupt the FMR1 gene. A large proportion of FXS patients have features of autism spectrum disorder (ASD). The protein encoded by FMR1, fragile X mental retardation protein (FMRP), binds mRNAs and regulates their translation. The identity and function of FMRP target mRNAs in human neurodevelopment and how they contribute to FXS are not well understood. In my preliminary data, parts of which formed the basis of my manuscript currently in press (Kwan et al., Cell, 2012), I show that nitric oxide synthase 1 (NOS1) mRNA is bound to and regulated by FMRP in the developing human neocortex and NOS1 protein is lost in fetal cases of FXS. Therefore, NOS1 loss of function represents a promising novel candidate mechanism of FXS. In this application, I propose to further pursue this exciting line of research. In Aim 1, I will examine how loss of NOS1 may contribute to the pathophysiology of FXS and ASD, with which FXS is often comorbid. Specifically, I will analyze: the neurobiological role of pyramidal NOS1 during development; the genetic contribution of NOS1, and genes that act upstream of NOS1 in the synthesis of nitric oxide (NO), to ASD; and the events downstream of NOS1 signaling in pyramidal neurons. For Aim 2, I propose to identify additional novel candidate mechanisms of FXS using postmortem human brain tissue and to test their dysregulation in FXS using FXS brains and neural stem cells. This multifaceted proposal is expected to provide training in several areas, including novel techniques in human genetics, biochemistry, human tissue processing, synapse analysis, neural stem cell culture, and high-throughput sequencing. This additional training is critical to my successful transition to independence. The progress of my training during the mentored phase and my search for an independent position will be overseen by my co-mentors and a committee of four additional faculty members with diverse expertise. The proposed studies have implications for current approaches to study of FXS and have the potential to uncover novel molecular pathways of not only FXS but also ASD.